The Role of Autophagy in Murine Cytomegalovirus Hepatitis.

Autophagy is involved in the pathogenesis of multiple pathogen infection. Previous studies have reported that human cytomegalovirus (CMV) activates autophagy in the early stage of infection and then inhibits autophagy. Little is known about the role of autophagy in murine CMV (MCMV) infection, especially in MCMV-induced hepatitis. The purpose of this study is to investigate the role of autophagy in MCMV hepatitis. BALB/c mice were infected with MCMV and a series of experiments involving western blot, immunofluorescence, immunohistochemistry, H&E (Hematoxylin and Eosin) staining and quantitative real-time polymerase chain reaction were performed in this study. The expression of SQSTM1/p62, PI3K, the ratio of phosphorylated Akt to total Akt, and the ratio of phosphorylated mammalian target of rapamycin (mTOR) to total mTOR were increased, and the expression of light-chain 3 (LC3)-II were decreased in the livers of infected mice on days 3 and 7 postinfection (p.i.). Compared with the untreated infected group, increased transcription level of MCMV glycoprotein B (gB), increased expression levels of interleukin1-ß (IL-1ß), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), decreased expression level of type I interferon α (IFN-α), as well as aggravated liver pathological injury were detected in starvation-treated infected group on days 3 and 7 p.i.; whereas decreased transcription level of MCMV gB, decreased expression levels of IL-1ß, AST and ALT, increased expression level of type I IFN-α, as well as alleviated liver pathological injury were detected in chloroquine (CQ)-treated infected group on day 3 p.i. In conclusion, autophagy is inhibited through activating the PI3K/Akt/mTOR pathway in the liver of BALB/c mice during MCMV infection, and autophagy may promote MCMV replication and aggravate liver pathological damage and inflammation. Further understanding of the interactions between autophagy and MCMV infection and its potential mechanism may bring new important cues to the control of MCMV infection and antiviral therapy.

Short-lived AIM2 Inflammasome Activation Relates to Chronic MCMV Infection in BALB/c Mice.

Absent in melanoma 2 (AIM2) inflammasome is a crucial link bridging the innate host defense and the subsequent adaptive immunity when activated by exogenous double stranded DNA (dsDNA). Through establishing models of disseminated murine cytomegalovirus (MCMV) infection in BALB/c and C57BL/6 mice, we evaluated dynamic expression of AIM2 inflammasome components and its relationship with pathological damage and viral replication, trying to figure out whether AIM2 inflammasome is related to the chronic mechanism of MCMV. BALB/c and C57BL/6 mice were sacrificed on day 0, 1, 3, 7, 14 and 28 post infection. Expression levels of AIM2, pro-caspase-1, caspase-1 p20, pro-IL1ß and mature IL1ß in primary peritoneal macrophages (PMs) and spleens were detected by Western blotting. Contents of IL18 in the serum were detected by ELISA. Pathological examinations of livers were performed, and mRNA levels of MCMV glycoprotein B (gB) in salivary glands also assessed. Results showed that expression levels of AIM2 in PMs and spleens of C57BL/6 mice increased on day 3, even continued to day 28; caspase-1 p20 and mature IL1ß increased on day 7, 14 and 28; the persistently high expression of IL18 in the serum started on day 1, showing a double peak curve. As for BALB/c mice, expression of AIM2 in PMs increased on day 1 and day 7, while contents of AIM2 in spleens increased on day 1 and day 3; caspase-1 p20 and mature IL1ß merely increased 7 days fter infection. Thereafter, expression levels of AIM2, caspase-1 p20, mature IL1ß and IL18 were limited; the duration of AIM2 inflammasome activation in BALB/c mice was much shorter than that in C57BL/6 mice. The severer pathological damage and more viral replications in BALB/c mice further proved the deficient antiviral immunity to MCMV. In conclusion, the activation of AIM2 inflammasome in BALB/c mice was short-lived, which is quite possibly related to the chronicity of MCMV infection.

Maternal Murine Cytomegalovirus Infection during Pregnancy Up-regulates the Gene Expression of Toll-like Receptor 2 and 4 in Placenta.

Increasing evidence has revealed that maternal cytomegalovirus (CMV) infection may be associated with neurodevelopmental disorders in offspring. Potential relevance between the placental inflammation and CMV-related autism has been reported by clinical observation. Meanwhile, abnormal expression of Toll-like receptor 2 (TLR2) and TLR4 in placenta of patients with chorioamnionitis was observed in multiple studies. IL-6 and IL-10 are two important maternal inflammatory mediators involved in neurodevelopmental disorders. To investigate whether murine CMV (MCMV) infection causes alterations in placental IL-6/10 and TLR2/4 levels, we analyzed the dynamic changes in gene expression of TLR2/4 and IL-6/10 in placentas following acute MCMV infection. Mouse model of acute MCMV infection during pregnancy was created, and pre-pregnant MCMV infected, lipopolysaccharide (LPS)-treated and uninfected mice were used as controls. At E13.5, E14.5 and E18.5, placentas and fetal brains were harvested and mRNA expression levels of placental TLR2/4 and IL-6/10 were analyzed. The results showed that after acute MCMV infection, the expression levels of placental TLR2/4 and IL-6 were elevated at E13.5, accompanied by obvious placental inflammation and reduction of placenta and fetal brain weights. However, LPS 50 µg/kg could decrease the EL-6 expression at E13.5 and E14.5. This suggests that acute MCMV infection during pregnancy could up-regulate the gene expression of TLR2/4 in placental trophoblasts and activate them to produce more proinflammatory cytokine IL-6. High dose of LPS stimulation (50 µg/kg) during pregnancy can lead to down-regulation of IL-6 levels in the late stage. Imbalance of IL-6 expression in placenta might be associated with the neurodevelopmental disorders in progeny.

Allium sativum-derived allitridin inhibits Treg amplification in cytomegalovirus infection.

This study investigated the effects of allitridin compound on murine cytomegalovirus (MCMV)-induced regulatory T cell (Treg; CD4(+) CD25(+) Foxp3(+) ) amplification in vivo and in vitro. One hundred twenty MCMV-infected mice were allocated at random into two groups for treatment with allitridin or placebo. Another 120 mock-infected mice were randomly allocated as controls for the allitridin treatment and placebo treatment groups. The mice were euthanized at various time points after infection (out to 120 days) to evaluate the effects of treatment on Treg presence and function, as well as MCMV infective load. Co-culture with mouse embryo fibroblasts (MEF) and MCMV was performed to evaluate allitridin-mediated Treg and anti-CMV effects. The maximum tolerance concentration (MTC) of allitridin was used to treat cells for 3 days. Changes in Foxp3 mRNA and protein levels, percentages of T cell subsets, and Treg-related cytokines (IL-10 and TGF-ß) were measured. Allitridin treatment did not influence Foxp3 expression and Treg proportion in uninfected mice, but did down-regulate each in infected mice during the chronic infection period. Additionally, allitridin treatment reduced the MCMV load in salivary glands. MTC allitridin treatment of co-cultures partially blocked MCMV induction of Foxp3 mRNA and protein expression. In vitro treatment with allitridin also increased significantly the percentages of Tc1, Tc2, and Th1, reduced the secreted levels of IL-10 and TGF-ß1, and significantly suppressed viral loads. In conclusion, allitridin can promote MCMV-induced Treg expansion and Treg-mediated anti-MCMV immunosuppression. Therefore, allitridin may be useful as a therapeutic agent to enhance the specific cellular immune responses against CMV.

[Immunopathological mechanism of spleen damage in acute disseminated MCMV infection].

AIM: To explore the immunopathological mechanism of spleen damage in acute disseminated infection of murine cytomegalovirus (MCMV) in vivo by observing the association of virus titers and the expressions of caspase-1 and pro-inflammatory factors (IL-1ß and IL-18) with the degree of pathological damage of the spleen. METHODS: BALB/c mice (n=24) were randomly divided into 2 groups (n=12 per group), experimental group and control group. The control mice were sham-infected. The experimental mice were infected with MCMV Smith for establishing the models of acute disseminated MCMV infection. Three mice of each group were randomly chosen to be killed on day 3, 7, 14 and 28 after infection, respectively. Viral titers in the spleen tissues were determined using a standard plaque assay; the expression of caspase-1 in the splenocytes was detected by Western blot; the expressions of IL-1ß and IL-18 in the spleen tissues were observed by immunohistochemical staining; the degree of spleen histological damage was observed by HE staining and graded by a semiquantitative method. RESULTS: Viral titers in the spleen peaked on day 3, but quickly diminished on day 7 and virus was not detected in the spleen on day 14 after infection. Compared with the normal control group, the protein levels of caspase-1 in MCMV-infected mice were markedly elevated on day 3 (P<0.01), and then dropped slowly; the expressions of IL-1ß and IL-18 in the spleen tissues gradually increased to the climax on day 7, then decreased on day 14 and turned to the normal on day 28. However, the pathological condition of the spleen in infected mice deteriorated gradually until day 14, and then showed obviously the signs of recovery on day 28. The spleen damage was aggravated following the elevation of IL-1ß and IL-18 expressions and alleviated after they declined. CONCLUSION: MCMV infection would stimulate the increase of caspase-1 expression and the production of pro-inflammatory factors (IL-1ß and IL-18). IL-1ß and IL-18 not only exert antiviral effect, but also might be involved in the immunopathological injury of spleen tissue during the acute disseminated MCMV infection.

Effects of allitridin on acute and chronic mouse cytomegalovirus infection.

This study investigated the effects of allitridin on acute and chronic mouse cytomegalovirus (MCMV) infections in vivo. The results demonstrated that allitridin reduced the titers of MCMV in salivary glands, and reductions in viral loads were confirmed by determining viral DNA and RNA levels in susceptible organs during the acute infection phase. Although allitridin did not eliminate MCMV, treatment reduced viral levels and facilitated healing of pathologic lesions in organs, particularly during the chronic infection phase. The results presented in this report suggest that allitridin could act as an effective agent against MCMV infections in vivo.

Identification of proteins that interact with murine cytomegalovirus early protein M112-113 in brain.

BACKGROUND: Murine cytomegalovirus (MCMV) early protein M112-113 is involved in viral DNA replication and believed to play a crucial role in the viral pathogenesis. To investigate the biological function of M112-113 protein in the pathogenesis of the brain disorders caused by cytomegalovirus (CMV), a screening for proteins interacting with M112-113 was performed by a yeast two-hybrid system. METHODS: Bait plasmid pGBKT7-M112-113 was constructed and transformed into AH109 yeast. After confirmation of the expression of MCMV M112-113 in yeast, the bait yeast was mated with a prey yeast containing mouse brain cDNA library plasmid to screen the proteins interacting with M112-113. Interactions between M112-113 and the obtained proteins were verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion. RESULTS: Two proteins interacting with M112-113 were identified, including metastasis-associated 1 (MTA1) and zinc finger, CCHC domain containing 18 (ZCCHC18). M112-113 protein could interact with MTA1 or ZCCHC18 in yeast and mammalian cells. CONCLUSION: The interactions of M112-113 with MTA1 or ZCCHC18 may be related to the pathogenesis of MCMV-associated disease in central nervous system.

[Role of allitridin in the blocking of murine cytomegalovirus induced regulatory T cells amplification in vitro].

OBJECTIVE: To investigate the effect of allitridin on murine cytomegalovirus (MCMV) induced regulatory T cells (Treg) amplification in vitro. METHODS: A co-culture system of T cells and MCMV infected mouse embryo fibroblasts (MEF) was established. A maximum tolerance concentration (MTC) of allitridin was added into the co-culture system. After 3 days, the change of Foxp3 mRNA was measured by real-time PCR. And the percentages of Tc1 (CD3(+)CD8(+)IFN-γ(+)), Tc2 (CD3(+)CD8(+)IL-4(+)), Th1 (CD3(+)CD8(-)IFN-γ(+)) and Th2 (CD3(+)CD8(-)IL-4(+)) were analyzed by flow cytometry. The production of IL-10 and TGF-ß in supernatants was detected with double-antibody sandwich ELISA while the viral load of culture quantified by plaque assay. All results were compared with those of the placebo group. RESULTS: The MTC of allitridin was 9.83 µg/ml in MEF. The treatment of 9.83 µg/ml allitridin could partly block the MCMV induction of Foxp3 mRNA expression [(87 ± 5) vs (114 ± 8), P < 0.01]. The percentages of Tc1, Tc2 and Th1 significantly increased to the levels of (12.42 ± 1.23)%, (4.28 ± 0.56)% and (13.25 ± 0.68)% respectively. They showed statistic differences with those of placebo controls [(6.85 ± 0.92)%, (2.34 ± 0.42)% and (9.32 ± 0.86)%; all P < 0.01]. Meanwhile, the levels of IL-10 and TGF-ß1 in supernatants also significantly decreased to (29.98 ± 3.15) pg/ml and (3.48 ± 0.23) ng/ml by allitridin treatment as compared with placebo controls [(38.21 ± 4.02) pg/ml and (5.31 ± 0.59) ng/ml, all P < 0.05]. In addition, the MCMV plaque assays showed that allitridin significantly suppressed the viral loads by one order of magnitude. CONCLUSION: Allitridin can partly retrieve MCMV-induced Treg expansion and Treg-mediated anti-MCMV immunosuppression so as to enhance the specific cellular immune responses against CMV.

CD4+CD25+ regulatory T cells suppress the immune responses of mouse embryo fibroblasts to murine cytomegalovirus infection.

Cytomegaloviruses (CMVs) cause common viral infectious diseases and are difficult for the host immune system to eliminate, which leads to persistent or chronic infection. To investigate the T cell immune response stimulated by murine cytomegalovirus (MCMV) infection and the role of CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs) in this process, T cells containing various proportions of Tregs were co-cultured with MCMV-infected mouse embryo fibroblasts (MEFs). MCMV infection stimulated proliferation of effector T cells as well as differentiation to Tregs, which consequently increased the expression of TGF-beta and IL-10. The proliferation of Tc1 (CD3(+)CD8(+)IFN-gamma(+)), Th1 (CD3(+)CD4(+)IFN-gamma(+)), and Tc2 (CD3(+)CD8(+)IL-4(+)) subsets was significantly suppressed with an increased proportion of Tregs in the co-culture system. Treg-depleted T cells inhibited viral load when co-cultured with MCMV-infected MEFs, however, this inhibitory effect was diminished when an increased proportion of Tregs was introduced. The suppressing effects of Tregs on effector T cells were attenuated by the addition of monoclonal antibody to TGF-beta, but not the one to IL-10, suggesting that TGF-beta is a major messenger involved in the immune suppressing effect of Tregs.